笋用竹林地有机农药污染土壤微生物修复机理研究
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摘要
在生态与食品安全备受全社会普遍关注的大背景下,本研究以浙江省主要产竹(县)市的竹林地土壤和竹笋为研究对象,对土壤和竹笋有机农药污染状况作了深入研究,并对残留状况作出了科学评价。鉴于竹林地土壤、竹笋存在有机农药残留的现状,针对性地开展了竹林地土壤有机农药污染微生物修复、修复效果影响因子及降解微生物分离鉴定等研究工作。研究结果如下:
1.竹林地土壤和竹笋有机农药残留现状
竹林地土壤中共检测到17种有机农药,其中,有机氯农药及其异构体11种,拟除虫菊酯类农药2种,有机磷农药4种。HCH检出率达100%,显著高于DDT(70.37%)。甲基对硫磷检出率96.30%,拟除虫菊类农药检出率较低(62.96%)。
土壤中检测出的有机农药中,除顺-氰戊菊酯残留量超过1000μg·kg-1外,其它种类农药的残留量均低于150μg·kg-1。HCH、DDT残留量均低于50μg·kg-1,有机磷农药最大残留量低于40μg·kg-1。这说明浙江省主要竹产区商品竹林土壤有机农药残留具有普遍性,有机氯、有机磷农药残留量较低,拟除虫菊酯类农药残留较高。
竹笋中检测到9种有机氯农药,有机磷农药和氨基甲酸酯类农药均低于检测限。HCH和DDT检测率均达100%,在竹笋中DDT更加稳定。五氯硝基苯检出率达75%。
竹笋检测出的有机氯农药残留量均低于100μg.kg-1,符合浙江省优质竹笋标准,但部分竹笋样品农药残留超过国家蔬菜水果标准(50μg.kg-1),其中,DDT超标率17.9%,HCH超标率3.6%。综合污染指数和系统聚类分析表明,试验笋样可以分为两类,即安全类(P<1),占82.14%,基本安全类(P=1～1.2),占17.86%。
商品竹林经营类型、经营水平和土地利用方式对竹林地土壤及竹笋有机农药残留有一定的影响,集约经营竹林显著高于粗放经营竹林,笋用竹林较毛竹笋材两用林高,山地竹林较原农业耕作地改种为竹林的低。
2.竹林地有机农药残留微生物原位修复技术
有机农药的原位微生物修复效果明显。使用菌剂15d内甲基对硫磷、丁硫呋喃丹、六六六三种有机农药降解率超过62%,分别达到83.08%、82.66%、62.21%,显著高于对照。雷竹林不同林分密度对甲基对硫磷微生物修复效果影响显著,在中密度下,既能获取较高的竹笋产量和经济产出,15d内甲基对硫磷降解率达84%以上。因此,应结合雷竹林丰产林分结构的建立,使竹林达到中密度水平,即立竹度12000-15000株·hm-1,再进行微生物修复。
在退化雷竹林土壤中共检测到3种有机磷农药,即乐果、甲基对硫磷、乙酰甲胺磷,且随着竹林退化程度的加剧残留量升高。采用有机磷农药降解菌剂进行田间微生物修复,15d内残留降解率超过50%,最高达100%。退化程度对降解率有显著影响,随着退化程度的加剧,降解率显著下降。
3.环境因子对微生物修复效果的影响
环境因子温度、光照、土壤pH值、土壤含水量对有机磷农药甲基对硫磷的微生物降解有着显著的影响。虽然随着时间的推移,甲基对硫磷的降解率逐渐增加,但降解速率逐渐降低。正交试验表明温度、土壤水分、土壤pH三因子在有机磷农药甲基对硫磷微生物降解过程中起到关键作用。获得最佳修复效果的环境因子组合为:温度25℃、光照60%、土壤含水率15%、土壤pH值6.03。
4.土壤养分元素含量对生物修复效果的影响
通过二次正交回归设计,确立了土壤主要养分元素含量与有机磷降解菌剂对甲基对硫磷农药降解效果的关系,推导出了二次方程,5d、10d、15d的回归方程均达显著水平,其中,5d时回归方程极显著。根据回归方程,推导出5d、10d、15d最佳降解率的土壤养分元素含量分别为:氮素(N)0.112 g·kg-1、0.113 g·kg-1、0.122 g·kg-1,磷素(P2O5)0.083 g·kg-1、0.096 g·kg-1、0.093 g·kg-1,钾素(K2O)0.077 g·kg-1、0.078 g·kg-1、0.088 g·kg-1。降解率分别为62.09%、84.79%和96.13%。
5.有机磷农药降解微生物分离
从取自化工厂排水口附近的土壤中分离出了2株有机磷农药降解菌,定名为OPDM-1、OPDM-2,二者均属于假单胞菌属(Pseudomonas sp.)。对目标菌株OPDM-2特性研究表明,菌株对有机磷农药具有较好的降解作用,液体培养条件下,72h菌株对100mg·L-1有机磷农药乙酰甲胺磷降解率达59.72%。
Now the environmental and food safety were concerned by all people, the pollution status of soil planted bamboo and its shoots ware investigated in Zhejiang province, and then the scientific evaluation were provided. Based the pesticide residue status, further research including bioremediation of organ-pesticides pollution, the role of environmental factor and isolation of degradation microorganism of organophosphorus pesticides were carried out. The main results were listed below:
1 The status of pesticide residue in soil and shoots
Seventeen organ-pesticides were detected in soil of bamboo forest, including eleven organochlorine pesticides and its isomers, four organophosphorus pesticides and two pyrethroids. The detection rate of HCH was up to 100%, which was higher than that of DDT. The detection of parathion–methyl was also up to 96.30%, and the detection rate of pyrethroid is low. The residual concentration of all was lower than 150μg·kg-1 excerpt esfenvalerate with the concentration of 1227.14μg·kg-1. The concentration of HCH and DDT was lower greatly than 50μg·kg-1, and that of organophosphorus pesticides was lower than 40μg·kg-1. So all that showed although the organ-pesticide residue in soil was ubiquitous, its concentration is low excerpt one pryethroid.
Nine organochlorine pesticides were detected in shoots, and none of organophosphorus pesticides, pyrethroids and others were detected. The detection rate of both HCH and DDT was up to 100%, and the stability of DDT was higher than that of HCH in shoos. The detection rate of pentachlornitrobenzene was 75%. The average of all organ-pesticide residue is lower than 50μg·kg-1. The Nemerow index and cluster showed that tested shoot samples can be divided into two types: safety shoot, accounting for 82.14%; and fundamental safety shoot, accounting for 17.86%. All indicated that organochlorine pesticides residue of bamboo shoots form Zhejiang province was universal, but not severe.
In addition, management level, management types and topographty influenced organ-pesticide residue in soil and shoots to some extent. The residual concentration of pesticides was higher in soil and shoots under intensive management bamboo forests, shoot-used bamboo forest, and bamboo forest converted from cropland than in those under extensive management bamboo forest, phyllostachys pubescens shoot-timber-used forests, and bamboo forest established on mountains, respectively.
2 Bioremediation of organ-pesticides residue in soil of bamboo forest
The result showed that pesticide concentration in soil can be decreased after bioremediation markedly. The degradation rate of parathion–methyl, carbofuran and HCH was higher than 60%, which was 83.08%, 82.66% and 62.21% respectively after 15-days bioremediation. The pesticide degradation rate of bioremediation was significantly higher than that of control. Stand density of bamboo plantation influenced bioremediation of parathion–methyl remarkably. Significantly difference at bioremediation was found among different density. The optimal density of bamboo plantation is 15000-18000 culms·hm-2.
Three organophosphorus pesticides including dimethoate, parathion–methyl, acephate were detected in soil of degenerative Phyllostachys praecox stand, and their residual concentration was positive correlation with degeneration. Bioremediation used by degradation microorganism can eliminate organophosphorus pesticides markedly. The degradation rate was higher than 69%, and up to 100% after 15-days bioremediation. Degeneration can influence bioremediation greatly. With the graveness of degeneration, the degradation rate decreased remarkably.
3 The influence of environmental factor on bioremediation
Environmental factors such as soil temperature, light, soil pH and water content of soil, influenced bioremediation of organophosphorus pesticides evidently. Although during the bioremediation, the degradation rate and content increased gradually, the degradation velocity decreased gradually. The result showed that soil temperature, soil pH and water content of soil were the key factors influencing on bioremediation of organophosphorus pesticides. The optimum factor combination of bioremediation was soil temperature 25℃, light 60%, soil pH 6.03 and water content of soil 15%.
4 The influence of fertilization on bioremediation.
The influence of fertilization on bioremediation was tested by quadratic orthogonal and regression method. The regression equation was established, and all reached significant level. Based on regression equation, the optimum fertilization combination was N-0.112g·kg-1, 0.113g·kg-1, 0.122g·kg-1; P2O5-0.083g·kg-1, 0.096g·kg-1, 0.093g·kg-1; K2O-0.077g·kg-1, 0.078g·kg-1, 0.088g·kg-1. At this condition, the degradation rate was up to 62.09%、84.79%and 96.13% at 5, 10 ,15 day after bioremediation respectively.
5 Isolation for microorganism degradation of organophosphorus pesticides
Two degraded microorganism were isolated from soil fetching from drainage outlet of Longyou insecticide factory and named OPDM-1、OPDM-2. They all were strains of Pseudomonas sp. Under laboratorial, OPDM-2 can degrade organophosphorus pesticides markedly.
1.竹林地土壤和竹笋有机农药残留现状
竹林地土壤中共检测到17种有机农药,其中,有机氯农药及其异构体11种,拟除虫菊酯类农药2种,有机磷农药4种。HCH检出率达100%,显著高于DDT(70.37%)。甲基对硫磷检出率96.30%,拟除虫菊类农药检出率较低(62.96%)。
土壤中检测出的有机农药中,除顺-氰戊菊酯残留量超过1000μg·kg-1外,其它种类农药的残留量均低于150μg·kg-1。HCH、DDT残留量均低于50μg·kg-1,有机磷农药最大残留量低于40μg·kg-1。这说明浙江省主要竹产区商品竹林土壤有机农药残留具有普遍性,有机氯、有机磷农药残留量较低,拟除虫菊酯类农药残留较高。
竹笋中检测到9种有机氯农药,有机磷农药和氨基甲酸酯类农药均低于检测限。HCH和DDT检测率均达100%,在竹笋中DDT更加稳定。五氯硝基苯检出率达75%。
竹笋检测出的有机氯农药残留量均低于100μg.kg-1,符合浙江省优质竹笋标准,但部分竹笋样品农药残留超过国家蔬菜水果标准(50μg.kg-1),其中,DDT超标率17.9%,HCH超标率3.6%。综合污染指数和系统聚类分析表明,试验笋样可以分为两类,即安全类(P<1),占82.14%,基本安全类(P=1～1.2),占17.86%。
商品竹林经营类型、经营水平和土地利用方式对竹林地土壤及竹笋有机农药残留有一定的影响,集约经营竹林显著高于粗放经营竹林,笋用竹林较毛竹笋材两用林高,山地竹林较原农业耕作地改种为竹林的低。
2.竹林地有机农药残留微生物原位修复技术
有机农药的原位微生物修复效果明显。使用菌剂15d内甲基对硫磷、丁硫呋喃丹、六六六三种有机农药降解率超过62%,分别达到83.08%、82.66%、62.21%,显著高于对照。雷竹林不同林分密度对甲基对硫磷微生物修复效果影响显著,在中密度下,既能获取较高的竹笋产量和经济产出,15d内甲基对硫磷降解率达84%以上。因此,应结合雷竹林丰产林分结构的建立,使竹林达到中密度水平,即立竹度12000-15000株·hm-1,再进行微生物修复。
在退化雷竹林土壤中共检测到3种有机磷农药,即乐果、甲基对硫磷、乙酰甲胺磷,且随着竹林退化程度的加剧残留量升高。采用有机磷农药降解菌剂进行田间微生物修复,15d内残留降解率超过50%,最高达100%。退化程度对降解率有显著影响,随着退化程度的加剧,降解率显著下降。
3.环境因子对微生物修复效果的影响
环境因子温度、光照、土壤pH值、土壤含水量对有机磷农药甲基对硫磷的微生物降解有着显著的影响。虽然随着时间的推移,甲基对硫磷的降解率逐渐增加,但降解速率逐渐降低。正交试验表明温度、土壤水分、土壤pH三因子在有机磷农药甲基对硫磷微生物降解过程中起到关键作用。获得最佳修复效果的环境因子组合为:温度25℃、光照60%、土壤含水率15%、土壤pH值6.03。
4.土壤养分元素含量对生物修复效果的影响
通过二次正交回归设计,确立了土壤主要养分元素含量与有机磷降解菌剂对甲基对硫磷农药降解效果的关系,推导出了二次方程,5d、10d、15d的回归方程均达显著水平,其中,5d时回归方程极显著。根据回归方程,推导出5d、10d、15d最佳降解率的土壤养分元素含量分别为:氮素(N)0.112 g·kg-1、0.113 g·kg-1、0.122 g·kg-1,磷素(P2O5)0.083 g·kg-1、0.096 g·kg-1、0.093 g·kg-1,钾素(K2O)0.077 g·kg-1、0.078 g·kg-1、0.088 g·kg-1。降解率分别为62.09%、84.79%和96.13%。
5.有机磷农药降解微生物分离
从取自化工厂排水口附近的土壤中分离出了2株有机磷农药降解菌,定名为OPDM-1、OPDM-2,二者均属于假单胞菌属(Pseudomonas sp.)。对目标菌株OPDM-2特性研究表明,菌株对有机磷农药具有较好的降解作用,液体培养条件下,72h菌株对100mg·L-1有机磷农药乙酰甲胺磷降解率达59.72%。
Now the environmental and food safety were concerned by all people, the pollution status of soil planted bamboo and its shoots ware investigated in Zhejiang province, and then the scientific evaluation were provided. Based the pesticide residue status, further research including bioremediation of organ-pesticides pollution, the role of environmental factor and isolation of degradation microorganism of organophosphorus pesticides were carried out. The main results were listed below:
1 The status of pesticide residue in soil and shoots
Seventeen organ-pesticides were detected in soil of bamboo forest, including eleven organochlorine pesticides and its isomers, four organophosphorus pesticides and two pyrethroids. The detection rate of HCH was up to 100%, which was higher than that of DDT. The detection of parathion–methyl was also up to 96.30%, and the detection rate of pyrethroid is low. The residual concentration of all was lower than 150μg·kg-1 excerpt esfenvalerate with the concentration of 1227.14μg·kg-1. The concentration of HCH and DDT was lower greatly than 50μg·kg-1, and that of organophosphorus pesticides was lower than 40μg·kg-1. So all that showed although the organ-pesticide residue in soil was ubiquitous, its concentration is low excerpt one pryethroid.
Nine organochlorine pesticides were detected in shoots, and none of organophosphorus pesticides, pyrethroids and others were detected. The detection rate of both HCH and DDT was up to 100%, and the stability of DDT was higher than that of HCH in shoos. The detection rate of pentachlornitrobenzene was 75%. The average of all organ-pesticide residue is lower than 50μg·kg-1. The Nemerow index and cluster showed that tested shoot samples can be divided into two types: safety shoot, accounting for 82.14%; and fundamental safety shoot, accounting for 17.86%. All indicated that organochlorine pesticides residue of bamboo shoots form Zhejiang province was universal, but not severe.
In addition, management level, management types and topographty influenced organ-pesticide residue in soil and shoots to some extent. The residual concentration of pesticides was higher in soil and shoots under intensive management bamboo forests, shoot-used bamboo forest, and bamboo forest converted from cropland than in those under extensive management bamboo forest, phyllostachys pubescens shoot-timber-used forests, and bamboo forest established on mountains, respectively.
2 Bioremediation of organ-pesticides residue in soil of bamboo forest
The result showed that pesticide concentration in soil can be decreased after bioremediation markedly. The degradation rate of parathion–methyl, carbofuran and HCH was higher than 60%, which was 83.08%, 82.66% and 62.21% respectively after 15-days bioremediation. The pesticide degradation rate of bioremediation was significantly higher than that of control. Stand density of bamboo plantation influenced bioremediation of parathion–methyl remarkably. Significantly difference at bioremediation was found among different density. The optimal density of bamboo plantation is 15000-18000 culms·hm-2.
Three organophosphorus pesticides including dimethoate, parathion–methyl, acephate were detected in soil of degenerative Phyllostachys praecox stand, and their residual concentration was positive correlation with degeneration. Bioremediation used by degradation microorganism can eliminate organophosphorus pesticides markedly. The degradation rate was higher than 69%, and up to 100% after 15-days bioremediation. Degeneration can influence bioremediation greatly. With the graveness of degeneration, the degradation rate decreased remarkably.
3 The influence of environmental factor on bioremediation
Environmental factors such as soil temperature, light, soil pH and water content of soil, influenced bioremediation of organophosphorus pesticides evidently. Although during the bioremediation, the degradation rate and content increased gradually, the degradation velocity decreased gradually. The result showed that soil temperature, soil pH and water content of soil were the key factors influencing on bioremediation of organophosphorus pesticides. The optimum factor combination of bioremediation was soil temperature 25℃, light 60%, soil pH 6.03 and water content of soil 15%.
4 The influence of fertilization on bioremediation.
The influence of fertilization on bioremediation was tested by quadratic orthogonal and regression method. The regression equation was established, and all reached significant level. Based on regression equation, the optimum fertilization combination was N-0.112g·kg-1, 0.113g·kg-1, 0.122g·kg-1; P2O5-0.083g·kg-1, 0.096g·kg-1, 0.093g·kg-1; K2O-0.077g·kg-1, 0.078g·kg-1, 0.088g·kg-1. At this condition, the degradation rate was up to 62.09%、84.79%and 96.13% at 5, 10 ,15 day after bioremediation respectively.
5 Isolation for microorganism degradation of organophosphorus pesticides
Two degraded microorganism were isolated from soil fetching from drainage outlet of Longyou insecticide factory and named OPDM-1、OPDM-2. They all were strains of Pseudomonas sp. Under laboratorial, OPDM-2 can degrade organophosphorus pesticides markedly.
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